Reagents and methods for classifying leukocytes

ABSTRACT

A reagent for classification of leukocytes includes (a) at least two cationic surfactants; (b) at least one organic compound bearing a hydrophobic group and an anionic group; (c) a buffer for adjusting pH into a range of approximately 2-8. Also disclosed is a method for classifying leukocytes with the reagent. With the reagent and method, erythrocytes are lysed rapidly and classification of leukocytes into five groups is achieved in the same channel. The reaction may be carried out at approximately between 10-40° C. and scattered light signals may be detected at two angles for measuring the classification of leukocytes into five groups.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese Patent Application No.200710125463.0, filed Dec. 24, 2007, for “REAGENTS AND METHODS FORCLASSIFYING LEUKOCYTES,” the disclosure of which is fully incorporatedherein by reference.

TECHNICAL FIELD

The present disclosure is related to reagents and methods forclassifying cells, and in particular, to reagents for classifying bloodleukocytes and methods for classifying blood leukocytes with thereagents.

BRIEF SUMMARY

A reagent for classifying leukocytes into five groups corresponding tolymphocytes, monocytes, neutrophils, eosinophils, and basophils isdisclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-3 show the classification of leukocytes from three blood samplesinto five groups with one embodiment of a reagent for classification ofleukocytes.

FIG. 4 shows the classification of leukocytes into five groups withanother embodiment of a reagent for classification of leukocytes.

FIG. 5 shows the classification of leukocytes into five groups with yetanother embodiment of a reagent for classification of leukocytes.

In FIGS. 1-5, 0 represents fragments, 1 represents lymphocytes, 2represents monocytes, 3 represents neutrophils, 4 representseosinophils, and 5 represents basophils.

DETAILED DESCRIPTION

In clinical testing fields, it is desirable to classify and countleukocytes in the whole blood of patients to support the diagnosis andtreatment of diseases.

Up to date, a number of devices and methods for classifying and countingleukocytes have been reported which classify and count leukocytes by thephysical, electrical and optical features of the treated cells after theblood has been treated with a lytic or a staining agent.

For example, some of these methods classify leukocytes by detecting thefluorescence emitted by the leukocytes stained with a staining agent,while others achieve the classification of leukocytes by detecting theelectrical signals or scattered light signals after the leukocytes havebeen particularly treated with a lytic agent.

CN 95115317.X discloses a method for classifying leukocytes into fivegroups with a reagent for classifying leukocytes into four groupstogether with a reagent for detecting basophils. The reagent forclassifying leukocytes into four groups comprises at least one ionicsurfactant, at least one negatively charged organic compound, a nonionicsurfactant, and a buffer. It is used to treat a first blood sample toclassify the blood cells into four groups: lymphocytes, monocytes,eosinophils, and basophils with neutrophils. The reagent for detectingbasophils is used to treat a second blood sample to classify thebasophils from others. By combining the results from both reagents, thismethod classifies leukocytes into five groups. The classification isdetected by a two-angle laser light scattering method.

U.S. Pat. No. 5,677,183 proposes a method for classifying leukocytesinto five groups and counting each group. The method employs a two-anglelight scattering method with a reagent for classifying leukocytes intofour groups and a reagent for detecting basophils. The reagent forclassifying leukocytes into four groups comprises at least one ionicsurfactant to lyse erythrocyte and damage the cell membrane ofleukocytes, at least one negatively charged organic compound to bindwith positively charged materials in the leukocytes to givemorphological changes to the leukocytes, a nonionic surfactant and abuffer to adjust the pH value. The reagent for detecting basophilscomprises at least one polyoxyethylene-type nonionic surfactant having apolymerization degree of 3-10, at least one cationic surfactant, and abuffer for adjusting the pH to 2.5-4.0. A first reagent is added to afirst blood sample to classify leukocytes into four groups: lymphocytes,monocytes, eosinophils, and basophils with neutrophils. A second reagentis added to a second blood sample to classify the basophils from otherleukocytes by morphological features and cell size information. Bycombining the results from both reagents, this method classifiesleukocytes into five groups.

U.S. Pat. No. 5,618,733 discloses a reagent for classifying leukocytesinto four groups comprising at least one ionic surfactant, eithercationic or anionic, to simultaneously lyse erythrocyte and damage thecell membrane of leukocytes; at least one organic compound having ananion to bind with the cationic component in the leukocytes to givemorphological differences among different groups of leukocytes; anonionic surfactant; and a buffer to adjust the pH value. Themeasurement may be carried out with a two-angle laser light scatteringmethod.

U.S. Pat. No. 5,747,343 proposes a method comprising: lysingerythrocytes in a short period of time with a reagent comprising atleast an anionic or ampholytic surfactant while maintaining the originalor approximately original morphology of leukocytes, and classifying theleukocytes into four groups by gathering the signals of both forwardscattered light and side scattered light.

U.S. Pat. No. 5,155,044 proposes a method and a reagent system for rapidisolation and analysis of leukocytes from a whole blood sample. Thehemolytic reagent system consists of a first reagent selected fromformic acid, acetic acid and combination thereof and a second quenchingreagent. The reagent lyses erythrocytes and creates subtle modificationsto the leukocytes to enable the classification into fivesub-populations. The advantage and uniqueness of this reagent system isthe surprising speed at which it lyses the erythrocytes and the abilityto further differentiate the leukocyte population. Following hemolysis,the quenching agent is added to retard further hemolysis and inhibit anyfurther dramatic changes to the leukocyte population. The treated bloodsample has retained its characteristic immunochemical response. Themeasurement may be carried out by a two-angle laser light scatteringmethod, or a direct current (DC) method and a radio frequency (RF)method, or a direct current (DC) method and a light scattering method.

U.S. Pat. No. 5,510,267 discloses a hemolytic reagent and a method forclassifying leukocytes into five groups. The hemolytic reagent comprisesan aromatic oxyethanol, an organic buffering agent having a pK value ofabout 8.5, and a nonionic detergent. The blood sample is first mixedwith a diluent solution, then the hemolytic reagent is added to lyse theerythrocytes while maintaining the light scattering features ofleukocytes intact for at least 30 seconds, so as to classify theleukocytes into five groups by collecting the signals of scattered lightat four angles.

CN 88101677.2 is a method for classifying leukocytes by lysing theerythrocytes with an acid lytic agent of pH 2.6-4 rapidly whilemaintaining the leukocytes intact and then followed by the addition of aquenching agent. The measurement may be carried out by light scatteringor a direct current/radio frequency method.

There are a number of problems with the conventional methods disclosed.For example, by a two-angle light scattering method, U.S. Pat. Nos.5,618,733 and 5,747,343 are capable of classifying leukocytes into fourgroups corresponding to lymphocytes, monocytes, neutrophils oreosinophils, but both fail to separate the basophils from others.

Additionally, CN 95115317.X and U.S. Pat. No. 5,677,183 achieve theclassification of leukocytes into five groups by combining the resultsobtained in two channels using two reagents to separately treat twoblood samples. U.S. Pat. No. 5,510,267 makes it possible to achieve theclassification of leukocytes into five groups in one channel bydetecting the scattered light signals at four angles, which complicatesthe equipment design and thus raises the equipment costs.

With regard to the classification techniques described in U.S. Pat. No.5,155,044 and CN 88101677.2, it is possible to classify leukocytes intofive groups in one channel merely at room temperature. With the changingof the ambient temperature, it is difficult to keep the temperatureconstant at 25° C. The measurements fluctuate with the temperature andthus hurt the measurement precision. Under the circumstances, athermostatic apparatus is required to eliminate the influence oftemperature change. In addition, to keep the temperature at 25° C., notonly heating devices but also cooling devices, are necessary,significantly increasing the equipment costs.

The present disclosure provides a reagent system as well as a method forprecisely classifying leukocytes into five groups within one channel ata broad range of temperature.

The present disclosure provides a reagent for classification ofleucocytes into five groups, wherein the reagent includes:

(a) at least two cationic surfactants capable of lysing erythrocytes andpartly damaging the cell membrane of leukocytes;

(b) at least one organic compound bearing a hydrophobic group and ananionic group capable of binding with a cationic component present inthe leukocytes to give morphological differences among the leukocytes;

(c) a buffer for adjusting pH into a range of approximately 2-8.

By the classification of leukocytes, it is intended to classifyleukocytes into five groups corresponding to lymphocytes, monocytes,neutrophils, eosinophils, and basophils, respectively.

In one embodiment, the cationic surfactants are quaternary ammoniumsalt-type cationic surfactants.

According to another specific embodiment, the cationic surfactants arequaternary ammonium salt-type cationic surfactants having a structurerepresented by the following formula:

wherein R1 is a C₆-C₁₆-alkyl or -alkenyl group, R2 and R3 eachindependently are a C₁-C₄-alkyl or -alkenyl group, R4 is a C₁-C₄-alkylor -alkenyl group or a benzyl group; and X is a halogen.

In one embodiment, the hydrophobic group on the organic compound isselected from an aromatic group, a hydrocarbon group having six or morecarbon atoms or a heterocyclic ring having more than 6 carbon atoms, andthe anionic group is selected from a carboxyl or sulfonate group.

In one embodiment, the organic compound is an acidic pigment.

The reagent may further include alcohols in an amount of approximately0.1-10% by volume of the reagent.

In one embodiment, the alcohol may be methanol, ethanol, isopropanol,n-butanol or 2-phenoxyethanol.

The present disclosure also provides a method for classifyingleukocytes. The method includes the steps of adding the above definedreagent to a blood sample and then measuring the cell size informationand the information on the morphological features.

Specifically, in one embodiment, the method comprises mixing the reagentfor classification of leukocytes with the blood sample at a ratio ofapproximately 10:1-100:1 for approximately 12-30 seconds at atemperature of approximately 10-40° C., and then measuring the cell sizeinformation with a low angle scattered light of approximately 1°-5° andmeasuring the information on the morphological features with a highangle scattered light of approximately 6°-20°.

The reagents and methods disclosed classify leukocytes into five groupsin the same channel, each group respectively corresponding tolymphocytes, monocytes, neutrophils, eosinophils, and basophils, whilsterythrocytes are lysed rapidly. With the reagents and methods disclosed,it is also suitable to carry out the classification of leukocytes notonly at room temperature, but also at a temperature in a broad range ofapproximately 10-40° C., significantly extending the range of applicabletemperature compared to conventional methods. On the other hand,conducting the classification above the room temperature not onlyimproves the classification reliability and stability by eliminating theinfluences of the environmental temperature, but also lowers theequipment costs through the omission of cooling devices. With thereagents and methods disclosed, the equipment costs may be furtherlowered since classification of leukocytes into five groups can beachieved through detection of the scattered light signals at merely twoangles.

One embodiment of a reagent for classification of leukocytes includes:

(a) at least two cationic surfactants capable of lysing erythrocytes andpartly damaging the cell membrane of leukocytes;

(b) at least one organic compound bearing a hydrophobic group and ananionic group, which is capable of binding with a cationic componentpresent in the leukocytes to give morphological differences among theleukocytes;

(c) a buffer for adjusting pH into a range of approximately 2-8.

The above reagent for classification of leukocytes into five groups canbe added into a whole blood sample to classify the leukocytes into fivegroups corresponding to lymphocytes, monocytes, neutrophils,eosinophils, and basophils and count each of the five groups bymeasuring the cell size information and the information on morphologicalfeatures.

In one embodiment, the cationic surfactants are quaternary ammoniumsalt-type cationic surfactants.

In some embodiments the cationic surfactants are quaternary ammoniumsalt-type cationic surfactants having a structure represented by thefollowing formula:

wherein R1 is a C₆-C₁₆-alkyl or -alkenyl group, R2 and R3 eachindependently are a C₁-C₄-alkyl or -alkenyl group, R4 is a C₁-C₄-alkylor -alkenyl group or a benzyl group; and X is a halogen.

The cationic surfactant may be used in an amount sufficient to lyseerythrocytes and to partly damage the cell membrane of leukocytes.Specifically, it is usually suitable to use approximately 100-10000mg/L, such as approximately 200-6000 mg/L, or alternatively,approximately 500-5000 mg/L cationic surfactant, although this amountcan be suitably modified depending upon the particular type ofsurfactant used. Some kinds of surfactants and the optimal amountsthereof are set forth in Table 1. Any combination of two or more ofthese surfactants may be used accordingly.

TABLE 1 Optimal amount of some surfactants Approximate OptimalSurfactants amount (mg/L) Octyltrimethyl ammonium chloride (OTAC, PTI)500-5000 mg/L Octyltrimethyl ammonium bromide (OTAB, PTI) 500-4500 mg/LDecyltrimethyl ammonium chloride (DTAC, PTI) 200-4000 mg/LDecyltrimethyl ammonium bromide (DTAB, PTI) 200-3000 mg/LLauryltrimethyl ammonium chloride (LTAC, PTI) 100-2000 mg/LLauryldimethylbenzyl ammonium chloride 100-2000 mg/L (DDBAC, PTI)Lauryltrimethyl ammonium bromide (LTAB, PTI) 100-2000 mg/LLauryldimethylethyl ammonium bromide 100-2000 mg/L (EDDAB, PTI)Lauryltriethyl ammonium bromide (DTEAB, PTI) 100-2000 mg/LMyristyltrimethyl ammonium chloride (MTAC, 50-1000 mg/L PTI)Myristyltrimethyl ammonium bromide (MTAB, 50-1000 mg/L PTI)Cetyltrimethyl ammonium chloride (CTAC, PTI) 50-500 mg/L Cetyltrimethylammonium bromide (CTAB, PTI) 50-500 mg/L

Any combination of surfactants can be used as long as the hemolyticactivity thereof is sufficient to create pores in the cell membrane ofleukocytes so as to make it possible for cytoplasm to overflow and anorganic compound to enter. The surfactants may be used in an amount farless than the amount for lysing the cell membrane so as to completelyexpose the cell nuclei. The hemolytic activity of the surfactant is inproportion to the chain length of R1. Typically, the more the carbonatoms, the higher hemolytic activity they will have.

In addition to the surfactants, the reagent may further contain anorganic compound capable of binding with the cations present in theleukocytes to give morphological differences among leukocyte categories.Such an organic compound bears a hydrophobic group (such as an aromaticgroup, a hydrocarbon group having six or more carbon atoms and aheterocyclic ring having more than 6 carbon atoms) and an anionic group(such as carboxyl, sulfonate group, and the like), and is negativelycharged in an aqueous solution and capable of binding with leukocytes tochange the morphological features of leukocytes. Almost all kinds ofacidic pigments can be used, such as Acid Blue series, Direct Blue, AcidGreen, Acid Yellow, Methyl Red, Methyl Orange, Aniline Blue, AlzarinYellow and the like. The amount used ranges from 10 to 1000 mg/L, suchas from 20 to 500 mg/L.

According to one embodiment, the reagent for classification ofleukocytes further contains a buffer for adjusting pH value. Typically,there is no particular limitation on the buffer. Any buffer systemcommonly used in the art, such as formate, phthalate, acetate,phosphate, TRIS, borate, carbonate, and the like, may be used. Thebuffer is typically used in an amount ranging from approximately 5-100mM to adjust the pH of the reagent within the range of approximately2-8.

Additionally, the reagent may further contain alcohol(s) as asolubilizer. There is no particular limitation on the types of thealcohols that may be used. Exemplary useful alcohols may be methanol,ethanol, isopropanol, n-butanol, 2-phenoxyethanol and the like. Theamount of the alcohols, as a solubilizer, typically ranges from 0.1% to10% by volume of the reagent.

An exemplary method for classifying leukocytes includes the steps of:measuring the cell size information and the information on themorphological features after the reagent has been added to the bloodsample and mixed for a period of time, and then classifying theleukocytes into five groups each corresponding to lymphocytes,monocytes, neutrophils, eosinophils, or basophils, and simultaneouslycounting each of the five groups of cells with a measuring system.

There is no particular limitation on the ratio between the reagent andthe blood sample. Typically, it is suitable that the ratio between theblood sample and the reagent is in a range of from approximately1:10-1:100, and the measurement may be carried out after they have beenmixed for approximately 12-30 seconds.

It is suitable to carry out the classification of leukocytes at atemperature in the range of approximately 10-40° C. As long as thetemperature is kept at a constant temperature within the range, thestability of the classification result for each test can be ensured. Insome embodiments it is desirable to keep the temperature at 35° C. sincesuch a temperature is above the room temperature, making it possible toprovide a constant temperature with only a heating device in thepractical application. The omission of a cooling device results in asignificant saving of the equipment costs.

According to the present disclosure, it is possible to classifyleukocytes into five groups within one channel. A laser detecting methodmay be used for the measurement of the cell size information and theinformation on the morphological features. Such a method can be carriedout by any devices known in the art, using low angle scattered light ofapproximately 1°-5° for measuring the cell size information and highangle scattered light of approximately 6°-20° for the information on themorphological features. An exemplary device is described in CN95115317.X, or any others known in the art can be used. The detection ofscattered light may also be performed with commonly used photodiodesensors.

The present disclosure will now be described in more detail withreference to the following examples.

EXAMPLE 1

A reagent for classifying leukocytes into five groups was prepared,which included:

Phthalic acid 1 g 2-phenoxyethanol 2.5 g Decyltrimethyl ammoniumchloride 3.5 g Octyltrimethyl ammonium bromide 1.75 g Toluidine blue0.025 g Water to 1 L pH 4.6

The pH value of the reagent according to the above formulation wasadjusted to 4.6.

1 mL of the above reagent was added into 10 μl blood at a temperaturemaintained at 35° C. The leukocytes were tested by a laser detectingmethod after being mixed for 15 seconds. Low angle scattered light ofapproximately 1°-5° was used to measure the cell size information andhigh angle scattered light of approximately 6°-20° was used to measurethe information on morphological features. The above reagent was used totreat three blood samples and the results are shown in FIGS. 1, 2 and 3,respectively. It is shown that the leukocytes are classified into fivegroups each corresponding to lymphocytes, monocytes, neutrophils,eosinophils, and basophils.

EXAMPLE 2

A reagent for classifying leukocytes into five groups was prepared,which included:

sodium dihydrogen phosphate 0.6 g Disodium phosphate 4.77 g Methanol 8 gLauryltrimethyl ammonium chloride 0.5 g Octyltrimethyl ammonium bromide3 g Acid Blue 0.05 g water to 1 L pH 7.5

The pH value of the reagent according to the above formulation wasadjusted to 7.5.

1 mL of the above reagent was added into 10 μl blood at a temperaturemaintained at 35° C. The leukocytes were tested by a laser detectingmethod after being mixed for 15 seconds. Low angle scattered light ofapproximately 1°-5° was used to measure the cell size information andhigh angle scattered light of approximately 6°-20° was used to measurethe information on morphological features. The result is shown in FIG.4, in which the leukocytes are classified into five groups eachcorresponding to lymphocytes, monocytes, neutrophils, eosinophils, andbasophils.

EXAMPLE 3

A reagent for classifying leukocytes into five groups was prepared,which included:

Formic acid 1.5 g Glycine 1 g Isopropanol 10 g Lauryldimethylbenzylammonium chloride 0.2 g Octyltrimethyl ammonium chloride 3 g Methyl red0.05 g water to 1 L pH 2.8

The pH value of the reagent according to the above formulation wasadjusted to 2.8.

1 mL of the above reagent was added into 10 μl blood at a temperaturemaintained at 35° C. The leukocytes were tested by a laser detectingmethod after being mixed for 15 seconds. Low angle scattered light ofapproximately 1°-5° was used to measure the cell size information andhigh angle scattered light of approximately 6°-20° was used to measurethe information on morphological features. The result is shown in FIG.5, in which the leukocytes are classified into five groups eachcorresponding to lymphocytes, monocytes, neutrophils, eosinophils, andbasophils.

While the present disclosure is described in detail with reference toparticular embodiments, the present invention is not to be construed inany case as being limited to these embodiments. It will be appreciatedthat various modifications may be made without departing from the spiritand scope of the invention and thus are considered as within the scopeof the present invention.

1. A reagent for classification of leukocytes, the reagent comprising:(a) at least two cationic surfactants capable of lysing erythrocytes andpartly damaging the cell membrane of leukocytes; (b) at least oneorganic compound bearing a hydrophobic group and an anionic groupcapable of binding with a cationic component present in the leukocytesto give morphological differences among the leukocytes; and (c) a bufferfor adjusting pH into a range of between approximately 2-8, wherein theat least two cationic surfactants are each independently quaternaryammonium salt-type cationic surfactants having a structure representedby the following formula

wherein R1 is a C₆-C₁₆-alkyl or -alkenyl group, R2 and R3 eachindependently are C₁-C₄-alkyl or -alkenyl group, R4 is a C₁-C₄-alkyl or-alkenyl group or a benzyl group, and X is a halogen; and wherein thereagent is configured to classify leukocytes into five groupscorresponding to lymphocytes, monocytes, neutrophils, eosinophils, andbasophils.
 2. The reagent for classification of leukocytes according toclaim 1, wherein the hydrophobic group on the organic compound isselected from: an aromatic group, a hydrocarbon group having six or morecarbon atoms or a heterocyclic ring having more than 6 carbon atoms; andthe anionic group on the organic compound is a carboxyl group or asulfonate group.
 3. The reagent for classification of leukocytesaccording to claim 1, wherein the organic compound is an acidic pigment.4. The reagent for classification of leukocytes according to claim 1,wherein the reagent comprises an alcohol in an amount of betweenapproximately 0.1-10% by volume of the reagent.
 5. The reagent forclassification of leukocytes according to claim 4, wherein the alcoholis chosen from at least one of: methanol, ethanol, isopropanol,n-butanol or 2-phenoxyethanol.
 6. The reagent for classification ofleukocytes according to claim 1, wherein R1 is a C₆-C₁₆-alkyl group, R2and R3 each independently is a C₁-C₄-alkyl group, R4 is a C₁-C₄-alkyl ora benzyl group; and X is a halogen.
 7. The reagent for classification ofleukocytes according to claim 6, wherein the cationic surfactants areselected from at least one of the following: octyltrimethyl ammoniumbromide, octyltrimethyl ammonium chloride, decyltrimethyl ammoniumchloride, laureltrimethyl ammonium chloride, and lauryldimethylbenzylammonium chloride.
 8. A method for classifying leukocytes, comprising:obtaining a reagent comprising: (a) at least two cationic surfactantscapable of lysing erythrocytes and partly damaging the cell membrane ofleukocytes; (b) at least one organic compound bearing a hydrophobicgroup and an anionic group capable of binding with a cationic componentpresent in the leukocytes to give morphological differences among theleukocytes; and (c) a buffer for adjusting pH into a range ofapproximately 2-8; mixing the reagent with a blood sample; measuringcell size information and information on morphological features of theleukocytes, and classifying the leukocytes into five groupscorresponding to lymphocytes, monocytes, neutrophils, eosinophils, andbasophils.
 9. The method according to claim 8, wherein mixing thereagent with the blood sample comprises: mixing the reagent and theblood sample at a ratio of approximately 10:1-100:1 for betweenapproximately 12-30 seconds at a temperature of between approximately10-40° C.
 10. The method according to claim 9, wherein measuring thecell size information comprises measuring cell size information with alow angle scattered light of approximately between 1°-5°; and whereinmeasuring the information on morphological features comprises measuringinformation on morphological features with a high angle scattered lightof approximately between 6°-20°.
 11. The method according to claim 8,wherein obtaining a reagent comprises obtaining a reagent that includescationic surfactants which are quaternary ammonium salt-type cationicsurfactants.
 12. The method according to claim 11, wherein the cationicsurfactants are quaternary ammonium salt-type cationic surfactantshaving a structure represented by the following formula:

wherein R1 is a C₆-C₁₆-alkyl or -alkenyl group, R2 and R3 eachindependently are a C₁-C₄-alkyl or -alkenyl group, R4 is a C₁-C₄-alkylor -alkenyl group or a benzyl group; and X is a halogen.
 13. The methodaccording to claim 12, wherein R1 is a C₆-C₁₆-alkyl group, R2 and R3each independently is a C₁-C₄-alkyl group, R4 is a C₁-C₄-alkyl or abenzyl group; and X is a halogen.
 14. The method according to claim 13,wherein the cationic surfactants are selected from at least one of thefollowing: octyltrimethyl ammonium bromide, octyltrimethyl ammoniumchloride, decyltrimethyl ammonium chloride, laureltrimethyl ammoniumchloride, and lauryldimethylbenzyl ammonium chloride.
 15. The methodaccording to claim 8, wherein the hydrophobic group on the organiccompound is selected from: an aromatic group, a hydrocarbon group havingsix or more carbon atoms or a heterocyclic ring having more than 6carbon atoms; and the anionic group on the organic compound is acarboxyl group or a sulfonate group.
 16. The method according to claim8, wherein the organic compound is an acidic pigment.
 17. The methodaccording to claim 8, wherein the reagent comprises an alcohol in anamount of approximately between 0.1-10% by volume of the reagent. 18.The method according to claim 17, wherein the alcohol is chosen from atleast one of: methanol, ethanol, isopropanol, n-butanol or2-phenoxyethanol.